Field of the Invention
Cystic fibrosis (CF) is the most common fatal genetic disease among persons of Caucasian origin. The frequency of the disease in this population is approximately 1 in 2500 live births Boat et al., 1989!, which translates into a carrier frequency of approximately 1 in 25. CF is associated with a wide-spread defect in the secretory processes of all secretory epithelia. Patients with CF, who rarely live for more than 30 years, exhibit abnormalities in a variety of respiratory, gastrointestinal and genitourinary tract systems, as well as elevated sweat electrolyte concentrations. Patients with CF exhibit abnormally viscid mucous secretions that block the airways and the pancreatic ducts. The blockage of the airways and pancreatic ducts are responsible for the two most clinically important manifestations of CF, that being chronic pulmonary infection and pancreatic insufficiency. The damage to the pancreas resulting in over 80% pancreatic insufficiency occurs in utero.
The above manifestations appear related to abnormal ion transport in the secretory epithelia of the affected organ Quinton, 1983; Knowles et al., 1983; Frizzell et al., 1986; Boucher et al., 1986; Quinton, 1990!. This was shown in the identification of reduced chloride permeability in isolated sweat ducts and nasal epithelia of patients with CF. This observation led to the conclusion that a fundamental defect in the transport of chloride (Cl) ions, and possibly other ions, across epithelial cells must exist.
The relative impermeability of epithelial cell membranes to Cl ions appears to be the primary defect in CF. The molecular basis (the gene) for this defect in Cl ion transport was mapped and identified in 1989 Riordan et al, 1989!. The protein product of the CF-associated gene is designated the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The CFTR protein is a single protein of approximately 170 kd and is made up of two repeated elements, each comprising six transmembrane segments and a nucleotide binding domain. The two repeats are separated by a large, polar, domain, designated R, containing multiple potential phosphorylation sites.
Normal CFTR protein in healthy individuals is found on the apical portion of epithelial cells which line the airway, gastrointestinal tract, and other ducts in the body. The CFTR chloride channel is activated by protein kinase A (PKA)-dependent phosphorylation and gated by the hydrolysis of ATP in two nucleotide binding domains, NBD1 and NBD2 Gadsby et al., 1995; Riordan et al, 1989!.
The most common mutation in CFTR responsible for CF disease is a deletion of three nucleotides that encode a single amino acid (phenylalanine) at amino acid position 508. This mutation is designated ".DELTA.F508," and is associated with approximately 70% of the cases of cystic fibrosis, the remaining 30% have mutations elsewhere. The .DELTA.F508 mutation results in an abnormal folding of the CFTR protein which is thought to be responsible for the improper localization of a large portion of the mutant .DELTA.F508 CFTR.
The mutations in the CFTR reduce the chloride channel activity Collins, 1992; Welsh et al., 1993!. Misfolded .DELTA.F508 is targeted for degradation in the endoplasmic reticulum; thus the predominant form of the .DELTA.F508-CFTR is decreases in the apical membrane Cheng et al., 1992!.
The mutant CFTR protein .DELTA.F508 CFTR has been shown to be synthesized in CF cells; however, a large part is retained in the cell where it appears to be rapidly degraded. Studies have shown that the .DELTA.F508 CFTR protein possesses functional characteristics similar to those of the normal CFTR protein in constructed lipid bilayers, but that a large portion simply does not reach the correct cellular location of the cell surface. Cheng et al., 1990; Denning et al. 1992, Li et al., 1993; Yang et al., 1993!. That portion of .DELTA.F508 CFTR which does reach the apical portion of the cell is not activated by drugs which activate protein kinase.
Current therapies for the treatment of CF include physical therapy, nutritional therapy and antibiotic therapy Ramsey, 1996!. These treatments are all directed toward treatment of the symptoms or effects of the disease and target the secondary effects of the disease; namely, obstructed airways, malnutrition, and chronic bacterial infections in the lungs. None of these approaches address the primary defect of the disease, the mutant CFTR protein and thereby the reduced chloride channel activity.
Recent discoveries have been used to attempt to treat CF including gene therapy and compounds which elevate cAMP levels. These approaches have not been successful. A new therapeutic modality or agent for the treatment of CF which is targeted to treating the underlying molecular dysfunction at the chloride channel level, would be highly desirable.
Furthermore, it would be useful to have additional therapeutic methods and compositions which can be used as drugs to restore the chloride channel function in cells with mutant CFTR, thereby providing a modality for treating Cystic Fibrosis at the level of restoring chloride channel function to mutant CFTR and which can be used in combination with other currently available therapeutics.